The present invention relates to a promoter of a gene expressed in renal cells. The promoter of this invention can be applied to the field such as gene therapy.
Sixty trillion various cells in a living body have essentially identical genomic DNA. For the normal physiological functions, the expression of these genes is strictly controlled through signals received by cell lines and cells. Therefore, elucidation of genes expressed specifically in each cell type is very important.
Mesangium is located in the center of lobula of capillary loop in glomerulus and is a tissue of a core that connects each lobule. Mesangium is covered by glomerular basal membrane and comprises mesangial cells which are separated from capillary cavity by endothelial cells and amorphous material (mesangial matrix) which is continuous with internal hyaline layer in glomerular basal membarane consisting of three layers.
A mesangial cell is known to play a pivotal role in maintaining the structure and function of a glomerulus and is considered to be the major cause of the onset of glomerular diseases such as glomerulonephritis and glomerulosclerosis. A mesangial cell is a target of disorders for each type of nephritis. For example, proliferation of mesangial cells and accumulation of extracellular mesangial matrix are thought to be the first step in which glomerulosclerosis is developed in a patient suffering from various glomerular diseases such as chronic glomerulonephritis and diabetic nephropathy, the two major causes of the end stage of renal failure [D. Schlondorff, Kidney Int., 49, 1583-1585 (1996); R. B. Sterzel et al., Glomerular mesangial cells. Immunologic Renal Diseases, pp595-626 (1997)]. Therefore, identification of genes expressed specifically in mesangial cells and elucidation of mechanism regulating its expression are helpful for understanding biological characteristics of mesangial cells and the causes of diseases relating to mesangial cells, and in turn, treating or diagnosing diseases relating to mesangial cells.
Through the determination of large-scale DNA sequences and through the database analysis, the present inventor isolated a gene named MEGSIN that is strongly expressed specifically in mesangial cells. The inventor also determined the whole nucleotide sequence of the gene and deduced the amino acid sequence of the novel protein (human MEGSIN) comprising 380 amino acids encoded by the whole cDNA clone of MEGSIN. Furthermore, the homology search in amino acid sequences with FASTA program using SwissProt database revealed that human MEGSIN belonged to SERPIN (serine protease inhibitor) superfamily [R. Carrell et al., Trends Biochem. Sci., 10, 20 (1985); R. Carrell et al., Cold Spring Harbor Symp. Quant. Biol., 52, 527 (1987); E. K. O. Kruithof et al., Blood, 86, 4007 (1995); J. Potempa et al., J. Biol. Chem., 269, 15957 (1994); E. Remold-O""Donnell, FEBS Lett., 315, 105 (1993)] [T. Miyata et al., J. Clin. Invest., 120, 828-836 (1998)].
Human MEGSIN is weakly expressed in human fibroblasts, smooth muscle cells, endothelial cells, and keratinocytes, but is strongly expressed in mesangial cells (that means human MEGSIN gene is expressed specifically in mesangial cells). When compared between IgA nephropathy patients or diabetic nephropathy patients and normal healthy people, the expression level of MEGSIN in renal tissue is significantly larger in IgA nephropathy patients or diabetic nephropathy patients [D. Suzuki et al., J. Am. Soc. Nephrol. 10, 2606-2613 (1999)]. Also, increase in expression level was observed in the model of mesangial proliferative glomerulonephritis using rats.
As indicated above, there is a possibility that the expression of MEGSIN gene is deeply involved in renal disease. Therefore, it is desired to reveal the actual condition of the regulatory mechanisms of the expression of MEGSIN gene, to clarify the function of human MEGSIN in vivo, and to provide useful promoter that is available for diagnosis and treatment of, for example, genetic disease caused by the mutation of MEGSIN or that expresses specifically in mesangial cells.
On the other hand, because the members of SERPIN superfamily to which human MEGSIN belongs are highly similar to each other in their primary structures, they are thought to be derived from the evolutionary common ancestral protein. Namely, as a result of the analysis of the phylogenetic tree constructed based on the number of mutated amino acids in the sequences [K. Suzuki et al., Tanpakushitsu Kakusan Koso, 34, 949-962 (1989)] and on the chromosomal gene structure [J. J. Bao et al., Biochem., 26, 7755 (1987)], it has been revealed that SERPIN superfamily has evolved through not less than 5 million years with various higher vertebrates. It is extreme characteristic of MEGSIN gene that it is expressed specifically in mesangial cells in glomerulus.
Recently, it was reported that genes of ion channels and genes involved in transportation are expressed specifically in kidney [S. J. Lolait et al., Nature, 357, 336-339 (1992); Y. Kanai et al., J. Clin. Invest., 93, 397-404 (1994); S. Uchida et al., J. Biol. Chem., 268, 3821-3824 (1993); S. Adachi et al., J. Biol. Chem., 269, 17677-17683 (1994); K. Fushimi et al., Nature, 361, 549-552 (1993); G. Gamba et al., J. Biol. Chem., 269, 17713-17722 (1994)].
However, these genes are located in epithelial cells of renal tubular and are not expressed in mesangial cells of glomeruli. Therefore, identification of the promoter and the transcription factors of MEGSIN gene can bring important information about the mechanism of gene expression depending on specific cell type. This information can also be applied to target cells in molecular genetics and gene transfer.
An objective of this invention is to provide a promoter of MEGSIN gene and its use.
The present inventor has determined nucleotide sequences of genomic DNA which is about 1.5 kb long including the upstream (5xe2x80x2 terminal) sequences of MEGSIN gene to reveal the regulatory mechanism of expression of MEGSIN gene. As a result of S1 nuclease protection assay, it has been revealed that four transcription initiation sites are existed in MEGSIN mRNA. It has also been revealed that the conserved transcription regulatory sequence that can be the transcription regulatory sites including AP1 binding site, cMyb binding site, and Oct-1 exist upstream of the transcription initiation site. Vectors in which various region of this transcription regulatory sequence were deleted and luciferase gene was integrated into the 3xe2x80x2 region have been constructed and the transcription regulatory region has been determined by detecting luciferase activity in cells transfected with the vector. As a result, two regulatory sequences have been identified that control transcription positively. The transcription regulatory activity of one promoter region that is located at 3xe2x80x2 end has been analyzed in detail by introducing nucleotide substitution based on the method of site specific mutagenesis. As a result, the present inventor has succeeded in determining the nucleotide sequence of the DNA that plays an important role in transcriptional regulation.
Therefore, the present invention relates to a promoter of MEGSIN gene and its use, and more specifically relates to the following:
(1) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a part thereof, the DNA having a promoter activity;
(2) a vector comprising the DNA of (1);
(3) the vector of (2), wherein a foreign gene is expressibly ligated downstream of the DNA of (1);,
(4) a cell transfected with the vector of (3);
(5) a method for screening a protein that binds to a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a part thereof, the method comprising the steps of: (a) contacting a test sample with the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a part thereof, and (b) selecting a protein that has an activity to bind the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a part thereof;
(6) a protein that can be isolated by the method of (5); and
(7) the protein of (6), wherein the protein is a transcription factor.
Herein, xe2x80x9cpromoterxe2x80x9d indicates the DNA region that exists near transcription initiation site and that controls the expression of a gene. In addition, xe2x80x9cpromoter activityxe2x80x9d indicates the activity of the promoter that controls the expression of a gene that exists downstream of the promoter.
The present invention provides a promoter of MEGSIN gene. As indicated in Example 3, the deletion or substitution of the nucleotide existing downstream of xe2x88x92128th position in the 5xe2x80x2 upstream region of MEGSIN gene often shows remarkable decrease in promoter activity. Thus, the promoter of this invention includes at least a part of 5xe2x80x2 upstream region of MEGSIN gene that is downstream of xe2x88x92128th position (SEQ ID NO: 1).
The promoter of this invention may have the nucleotide sequence of SEQ ID NO: 1 in which one or more nucleotides are substituted, as long as it includes at least a part of the region downstream of xe2x88x92128th position (SEQ ID NO: 1) and as long as it has a promoter activity. However, as indicated FIG. 7, mutations in the downstream region of xe2x88x92128th position: m1 mutation (nucleotide substitutions at xe2x88x92128th and xe2x88x92127th positions), m2 mutation (nucleotide substitutions at xe2x88x92120th, xe2x88x92118th, and xe2x88x92117th positions), m3 mutation (nucleotide substitutions at xe2x88x92116th and xe2x88x92115th positions), m4 mutation (nucleotide substitutions at xe2x88x92113th and xe2x88x92112th positions), m5 mutation (nucleotide substitutions at xe2x88x92106th and xe2x88x92105th positions), m6 mutation (nucleotide substitutions at xe2x88x92100th and xe2x88x9298th positions), and m7 mutation (nucleotide substitutions at xe2x88x9294th and xe2x88x9293rd positions) cause the decline of promoter activity.
Therefore, substitutions of these nucleotides are not preferred when the high promoter activity is required.
The promoter of this invention can be isolated by the method such as screening of a genomic DNA library. That is, the promoter can be isolated by hybridization screening of a genomic DNA library of human or other animals using the known MEGSIN cDNA or a part thereof as a probe or by polymerase chain reaction (PCR) using the primers that is designed based on the sequence of MEGSIN cDNA or MEGSIN genomic DNA and using a genomic DNA library of human or other animals as a template.
The promoter of this invention can also be produced by following the standard methods using chemical synthesis of nucleic acids, such as phosphoamidide method [Mattencci, M. D. and Caruthers, M. H. J. Am. Chem. Soc. 103, 3185 (1981)] and phosphite triester method [Hunkapiller, M. et al., Nature 310, 105 (1984)].
The promoter region and the enhancer region (existing in an intron or 3xe2x80x2 noncoding region and including the DNA region that promotes the expression of the gene) of MEGSIN gene existing in the DNA fragments can be obtained, for example, by the same method described in Unexamined Published Japanese Patent Application (JP-A) No. Hei 6-181767 or in the reference [The Journal of Immunology (1995) 155, 2477-2486, Proc. Natl. Acad. Sci. USA (1995) 92, 3561-3565].
In general, the presence or the strength of the promoter activity can be judged by expressibly ligating, downstream of an candidate promoter, the gene (reporter gene) encoding the protein that can easily determined quantitatively by, for example, color or luminescent reaction, by transfecting a host cell with it, and by detecting the color or luminescent reaction.
Specifically, a promoter region can be obtained by the following method but not limited thereto.
1) DNA including transcription regulatory region from genomic DNA or a genomic library as indicated above is cloned.
2) MEGSIN gene is digested with restriction enzyme to obtain a DNA containing a translation initiation codon of MEGSIN gene and comprising the promoter region (2 to 5 kbp) upstream thereof, and to determine the nucleotide sequence. The transcription initiation site (+1) is determined using, as a template, poly(A)+ RNA prepared from mesangial cells and such by the primer elongation method using primer DNA selected from cDNA sequence at 5xe2x80x2 end site of MEGSIN gene. A site possibly comprising the promoter activity is predicted by searching transcription factor binding sequence from the nucleotide sequence.
3) The DNA fragment excluding the coding region of MEGSIN gene from the DNA obtained in 2) is subcloned in a plasmid, and reporter gene (for example, chloramphenicol acetyl transferase (CAT) gene or a luciferase gene etc.) is ligated downstream of the 2 to 5 kbp DNA fragment to construct a reporter plasmid. Similarly, DNA fragments corresponding to various sites upstream of MEGSIN gene, in which 5xe2x80x2 and 3xe2x80x2 end sites are stepwise removed, are prepared by digestion with restriction enzymes or by PCR and such to include possible promoter regions. A reporter gene is ligated downstream of these DNA fragments to construct a reporter plasmid. A DNA fragment in which one or more nucleotides are appropriately replaced, deleted, added, and/or inserted by site specific mutagenesis is prepared. A reporter plasmid is constructed in which a reporter gene is ligated downstream of the DNA fragment.
4) A promoter region existing at the upstream region of MEGSIN gene is identified by measuring the reporter activity (for example, CAT activity or luciferase activity etc.) in animal cells that is transformed with the reporter plasmid constructed in 3).
An enhancer region existing in 3xe2x80x2 noncoding region or an intron can be identified by, for example, screening, as a probe, a genomic library with the MEGSIN cDNA and such, cloning MEGSIN genomic DNA, and conducting the experiment in the same method as that in the case of the promoter above.
A promoter of this invention has an activity to highly express a gene ligated downstream thereof in kidney (mesangial cells). Thus, the promoter of this invention can be used, for example, to develop a vector that can control the expression of desired genes in kidney. The promoter of this invention can also be expected to have the same effect in other cells (or organs) that has transcription factors that activate the promoter of this invention. Such promoter as is activated specifically in kidney can be used, for example, for constructing vectors for gene therapy of renal disease.
When the promoter of this invention is used for the kidney-specific expression of a gene, desired gene to be expressed in kidney is expressibly ligated downstream of the promoter, and is introduced into a target cell. The phrase xe2x80x9cexpressibly ligatedxe2x80x9d indicates that the gene is ligated to the promoter of this invention so that its expression is possible.
When a promoter of this invention and a gene controlled by the promoter are introduced into a target cell, they can be integrated into any appropriate vectors. The promoter of this invention can be used alone or in association with transcription regulatory sequences such as enhancer and silencer.
Vectors derived from, for example, retrovirus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus, bovine papillomavirus, adenovirus, adeno associated virus, sindbis virus, and poxvirus can be used for gene therapy. Formulation of liposome such as thermosensitive liposome, blood stable liposome, cationic liposome, pH dependent liposome, rearranged liposome including a envelope protein of virus, and such; formulation of membrane fusion liposome, which has membrane fusion ability of virus, such as HVJ (Sendai virus)-liposome [T. Nakagawa et al., Drug Delivery System, 11, 411 (1996)], VSV (vesicular stomatitis virus)-liposome (Japanese Patent Application No. Hei 9-357506), and such; and so on are also available.
Target cells into which the vectors are introduced can be, for example, mesangial cells, renal tubule cells, macrophage, lymphocyte, endothelial cells, and tumor cells.
Besides the methods described above, general gene manipulations for preparing the promoter of this invention and recombinant vector having the promoter and for transfecting a cell with the vector and such can be conducted by following, for example, the standard methods described in xe2x80x9cMolecular Cloningxe2x80x94A Laboratory Manualxe2x80x9d (Cold Spring Harbor Laboratory, N.Y.).
Because mutations in the promoter of this invention may cause serious genetic disease, the promoter is expectedly applicable to gene diagnosis. This gene diagnosis can be achieved by, for example, detecting the mutation by direct sequencing using the method such as single strand conformational polymorphism, DNA fingerprinting method, and PCR method, by mutation analysis using gene-specific oligonucleotide probe, etc.
Because human MEGSIN belongs to SERPIN superfamily, the disorder of human MEGSIN can cause thromboembolism by promoting the ability of blood clotting and hemorrhagic disease by promoting the ability of fibrinolysis [Suzuki et al., Tanpakushitsu Kakusan Kouso, 34, 949-962 (1989)]. This suggests that drugs affecting the transcription activity of the promoter of this invention can act on onset or repression of these diseases. Therefore, the promoter of this invention can be used for the screening of drugs for these diseases.
Because the expression level of MEGSIN is promoted in IgA nephropathy patients and diabetic nephropathy patients, it is thought to be involved in onset of renal disease. Therefore, onset or promotion of IgA nephropathy and diabetic nephropathy can be repressed by administering, to the patients, drugs that control the promoter activity of this invention.
This invention relates to the method for screening a protein that binds to the promoter of this invention. A protein that binds to the promoter of this invention is, for example, a transcription factor. xe2x80x9cA transcription factorxe2x80x9d indicates a protein that binds to the promoter of this invention and regulates positively or negatively the expression of a gene existing downstream of the promoter.
The screening method of this invention includes the steps of: (a) contacting a test sample with the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or part thereof and (b) selecting a protein that has an activity to bind the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a part thereof.
The screening method of this invention can be conducted by the known method to one skilled in the art [referred to Shin Saibou Kougaku Jikken Purotokoru (New Cell Engineering Experiment Protocol), Shujun-sha; Biomanual series 5 Tensha Inshi Kenkyu-hou (method for studying transcription factors), Yodo-sha; and DNA and Cell Biology, 13, 731-742 (1994)], for example, a method using affinity column, Southwestern method, footprinting method, gel shift method, and one-hybrid method.
The affinity column method can be performed by applying a nucleic extract onto an column in which the promoter of this invention obtained in the above manner are immobilized on Sepharose or latex beads, washing the column, and eluting the binding transcription factor using a DNA comprising the same sequence as that immobilized in the column.
In the case of Southwestern method, for example, cDNA is constructed from mRNA derived from the cells in which a transcription factor that binds to the promoter of this invention is expected to be expressed (for example, mesangial cells). Then, the cDNA is integrated into an E. coli expression vector, such as xcexgt11, to construct a cDNA library, and a fusion protein with xcex2-galactosidase is synthesized. The fusion protein is adsorbed on a nitrocellulose membrane, and a phage that synthesizes the fusion protein showing binding activities is selected using, as a probe, a radiolabeled DNA fragment of the promoter of this invention.
In the case of footprinting method, DNA sequence that binds to protein can be determined by using radiolabeled promoter as probe, mixed it with a nuclear extract, digesting it by DNase I, and conducting electrophoresis.
In the case of gel shift method, a probe is constructed from a sequence in the promoter region, and radiolabeled. Then, the radiolabeled probe and a nuclear extract are mixed, and electrophoresed to determine presence or absence of a nuclear protein that binds to the probe.
In the case of one-hybrid method, for example, sequence that contains tandem repeats of at least 3 copies of MEGSIN promoter sequence are ligated upstream of the reporter gene, and then integrated into the yeast genome to construct reporter strain. The above cDNA is ligated to the coding region for activation domain of GAL4 (transcription activating factor binding to DNA of yeast) (GAL4 AD), and the activation domain (AD) libraries that encode this fusion protein are constructed and introduced into the reporter strain above. Binding of the hybrid protein of AD and a DNA binding protein that binds to the MEGSIN promoter sequence activates the transcription. The effect can be detected by the expression of the reporter gene.